The basic objectives of this component will continue to utilize genetic strategies to identify and characterize genes of major significance in the development of human tumors as well as the response of tumors to treatment. The main focus of this program has been and continues to be the process of tumorigenesis in nephroblastoma (Wilms tumor). We will continue to study the molecular basis of the action of the Wilms tumor suppressor gene, WT1, discovered and characterized during the previous granting period. We will extend our studies involving gene targeting of mouse ES cells in order to understand the role of the WT1 gene in tumorigenesis and normal development as well as to identify the functional roles of alternatively spliced forms of the WT1 polypeptide. We will continue the biochemical and functional characterization of WT1 by extending our work on interaction partners for the WT1 polypeptide using the yeast two hybrid system, to characterize WT1 interactions with other polypeptides using immunochemistry, to develop cell based systems to directly demonstrate the effects of WT1 on specific target genes and to utilize in vitro transcription and splicing systems for the alternate forms of the WT1 polypeptide. To gain deeper insight into WT1 function, we will explore the role of WT1 in systems other than kidney such as hematopoiesis. We will complete identification and characterization of the WT2 tumor suppressor gene using positional cloning strategies and analysis of mutations and deletions in human tumors. We will further analyze the role of apoptosis in tumor treatment response using genetically based approaches such as further study of the tumor cells deficient for p53. These research strategies will provide the framework for gene identification and characterization extending beyond the immediate province of Wilms tumor to other tumor types which share common mechanisms of tumorigenesis with Wilms tumor as well as to further investigate the underlying basis of response to chemotherapeutic agents and radiation using extensions of the genetic approaches developed during the previous grant period.